A powerful platform technology for the discovery and development of new medicinal drug is macromolecule intracellular transduction technology (MITT) enabled with cell-penetrating peptides (CPPs) that provide cell-permeability of macromolecules in vitro and in vivo. A common problem with small molecules is the potential for off-target drug interactions. In addition, a limitation of macromolecules is the fact that proteins and nucleic acids are unable to be intracellularly delivered. To address these issues, MITT provides an improved method to deliver biologically active macromolecules including therapeutic proteins into cultured cells and animal tissues.
Plasma membrane normally acts as an impermeable barrier to constrain cellular internalization of macromolecules, such as oligonucleotides, DNA, RNA, peptides and proteins. Numerous difficulties have restricted the delivery of these macromolecules to a desired target: poor penetration into a cell and/or tissue; toxicity when delivered systemically due to the insufficient specificity of targeting to a particular cell and/or tissue; degradation in which limited amounts are delivered to the targeted region that may result in undesirable side effects; and side effects when delivered in a high concentration in order to attain a sufficient local concentration at a certain target cell and/or tissue. In order to address these problems, several carrier-mediated delivery systems have been developed. Latest developments have involved the use of peptide-based delivery systems. The use of hydrophobic CPPs has several advantages including various peptide sequence modification. This enables the engineering of carriers that can enter different cellular subdomains and/or are able to relocate various types of cargo molecules.
In principle, protein-based therapeutics offers a way to control biochemical processes in living cells under non-steady state conditions and with fewer off-target effects than conventional small molecule therapeutics. However, systemic protein delivery in animals has been proven difficult due to poor tissue penetration and rapid clearance. Intracellular macromolecule transduction exploits the ability of various CPPs such as specific basic, amphipathic, and hydrophobic peptide sequences to enhance the penetration of proteins and other macromolecules by mammalian cells. Although intracellular macromolecule transduction has been widely used, systemic delivery of proteins in animals has been proven difficult due to inefficient cytoplasmic delivery of internalized proteins and poor tissue penetration. This problem had been especially true for cationic protein transduction domains (PTDs, e.g. HIV Tat, Hph-1, antennapedia, polyarginine, etc.), where the predominant mechanisms of protein uptake—absorptive endocytosis and macropinocytosis—sequester significant amounts of protein into membrane-bound and endosomal compartments, thus limiting protein bioavailability. Chimeric CPPs containing mixed types of sequences such as hydrophilic, basic and hydrophobic amino acids have been revealed to have toxicity, thus this type of CPPs has been restricted from its usage. Greater success has been reported for a sequence such as membrane translocating sequence (MTS) or membrane translocating motif (MTM) derived from the hydrophobic signal peptide of fibroblast growth factor 4 (FGF4). The MTS/MTM has been used to deliver biologically active peptides and proteins systemically in animals (in particular to liver, lung, pancreas and lymphoid tissues), with dramatic protection against lethal inflammatory disease and pulmonary metastases.
Previously, hydrophobic CPPs (MTS/MTM) or macromolecule transduction domain (MTD) have been reported. However, many efforts to develop cell-permeable therapeutic proteins by using these reference hydrophobic CPP sequences have been hampered by poor solubility of the recombinant proteins in physiological buffer condition and relatively low cell-permeability for further clinical development and application. Although there has been a consensus that hydrophobic CPP-dependent uptake of protein cargo is a powerful way for developing protein-based biotherapeutics, further improvements are required to solve the critical problems influenced by non-cargo specific factors such as protein aggregation, low solubility/yield, and poor cell/tissue-permeability of the recombinant CPP-fused proteins. These CPPs have non-common sequence and non-homologous structure of the sequences.